The goal of this research is to define and characterize the neural networks governing two sequentially-linked behavioral programs in the fly. The first is an adaptive, environmentally-sensitive program that mediates the search for safe surroundings, and the second is a hormonally-driven program that serves to expand the recently developed wings of the newly emerged adult. The first program is used to find a safe perch from which the immobile fly can expand its wings, and the second program initiates expansion. Because wing expansion must be undertaken within several hours of emergence, the need for safety must be balanced by the imperative to expand and each individual fly must decide when (and under what environmental circumstances) to expand. Wing expansion thus provides a behavioral paradigm for studying decision-making, the most fundamental aspect of behavioral integration, and for understanding how hormonal and environmental factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Such understanding should, in turn, shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. In our effort to understand the behavioral program that governs wing expansion, a particular priority has been to characterize the neural network that controls this process. In a series of papers over the last several years, we have systematically defined the functional roles of neurons within one component of the network: a group of approximately 50 peptidergic neurons that act as a command system for wing expansion. As we have shown previously, suppression of these 50 neurons blocks expansion (Luan et al., J. Neurosci., 2006) while their activation is sufficient to induce that process (Peabody et al., J. Neurosci., 2009). In addition, we have demonstrated the essential contribution to wing expansion of a subset of 16 neurons within this command system that secrete the hormone bursicon (Peabody et al., J. Neurosci., 2008). Over the past year, we have extended our functional characterization of the bursicon-expressing neurons, and have reported our findings in a paper published in the Journal of Neuroscience (Luan et al., 2012). As reported in that paper, stimulation of the bursicon-expressing neurons elicits the entire wing expansion motor program in newly emerged adult flies. The bursicon-expressing subset of neurons thus shares the command capability of the larger group of peptidergic neurons. More remarkably, we report that single pair of bursicon-expressing neurons located in the subesophageal ganglion (which we call the BSEG) also has this capability and can drive the entire wing expansion program when stimulated. These two neurons thus act as command neurons for wing expansion. This finding was made possible by combining our previously developed UAS-TRPM8 technique for neuronal activation (Peabody et al., J. Neurosci., 2009) with our Split Gal4 system for refined transgene targeting (Luan et al., Neuron, 2006). By using the Split Gal4 system in conjunction with suppressors of excitability we were also able to characterize other functional properties of the BSEG neurons. Indeed, targeted neuronal silencing of the BSEG showed their essential contribution to wing expansion behavior, in that flies in which the BSEG were suppressed could no longer perform the motor patterns that normally underlie wing expansion. In fact, silencing the BSEG abrogated the flies' ability to expand their wings under almost all conditions. Surprisingly, under one condition, namely in the complete absence of environmental perturbation, flies lacking BSEG function were able to expand. In this case, however, they used an alternate set of motor patterns to do so. Our findings thus provide an interesting example of behavioral compensation, in which the loss of a preferred behavioral strategy is compensated by the recruitment of an alternative strategy so that an essential process can be completed. Such compensation is commonplace in response to brain injury, and may also come into play when genetic lesions disrupt the normal development of brain circuits, as occurs in mental disorders such as autism and schizophrenia. Understanding the mechanisms that govern compensatory changes--and understanding how they may fail--is thus potentially important for understanding how the nervous system responds to injury or developmental aberration. Because the wing expansion circuit is experimentally tractable, we are now in a position to ask precisely how circuits are organized and/or altered to permit such compensation to occur. A second focus of our characterization of the command system for wing expansion involves identifying its downstream cellular effectors. During the past year, we have succeeded in identifying neurons that express the bursicon receptor, which is encoded by the rickets gene. To do so, we developed a novel strategy that exploits the self-cleaving T2A peptide to co-express the Gal4 transcription factor in cells that express rickets. The resulting rickets-Gal4 driver line has allowed us to identify rickets-expressing neurons by driving the expression of the reporter, UAS-EGFP, and also to confirm their identity by driving a UAS-rickets transgene to rescue the behavioral deficits of rickets null mutants. These results were presented in a paper published earlier this year in the journal Genetics (Diao & White, 2012), which described the T2A-Gal4-In-Frame-Fusion strategy we used to make rickets-Gal4. We anticipate that this strategy will be of very general utility to other Drosophila researchers and are currently expanding the T2A-Gal4-In-Frame-Fusion toolkit so that we and others can fully exploit this strategy. In other on-going work, we are using the rickets-Gal4 driver to characterize the functional roles of subsets of rickets-expressing neurons in the wing expansion circuit. In this work, we are exploiting a tool for refined transgene targeting, similar to our earlier Split Gal4 technique, called the Split LexA system. This system, which we developed in collaboration with the laboratory of Dr. Chi-hon Lee (Ting et al., Genetics, 2011), allows the expression patterns of Gal4 driver lines, such as rickets-Gal4, to be parsed into subpatterns. In this way, subsets of rickets-expressing neurons can be independently manipulated to understand their functions. In summary, we have made substantial progress in functionally characterizing essential components of the circuit that governs post-emergence behavior in Drosophila. In addition, we have continued to develop useful tools for circuit mapping that can be used not only by us, but also by other members of the Drosophila research community. As we use these tools to extend and refine our analysis of the wing expansion circuit, our work should provide insight into the principles used by all nervous systems to generate and organize behavior. In generating a functional map of the wing expansion circuit, we will also position ourselves to initiate an analysis of its developmental determinants at the level of individual cells and cell-fate decisions by leveraging the powerful molecular genetic tools available for studying development in Drosophila.